A flexible system has been created and characterized which allows the semisynthetic assembly of novel tRNA genes and then the in vivo measurement of the maturation, modification, aminoacylation, and overall ribosomal function of the new gene products. Experimentation with this system, and a study of natural tRNA sequences, has yielded a model for anticodon loop and stem structural requirements for efficient tRNA function at the ribosomal A site. This grant proposes to extend this previous work by systematically testing the anticodon structure-function model by synthesis of previously "impossible" tRNA suppressors, by expanding the technique to other sections of the tRNA molecule, and by use of the method on translators of other codons. Furthermore, the product of this synthetic work is a large series of homologous tRNA's varying in structure, position-by-position, in a way never previously achieved. This homologous series can also be applied to detailed in vitro biochemical studies on the relation of structure to tRNA function in each of the elementary steps of genetic translation.